In vivo drug metabolism is performed by cytochrome P450 (which, hereafter, may be referred to as “P450”), which is mainly present in the liver. P450 constitutes a superfamily comprising many genes. P450 genes having amino acid sequence homology of higher than 40% are classified as belonging to the same family, and those exhibiting 55% or higher amino acid sequence homology within the same family are classified as belonging to its subfamily (Nelson et al., Pharmacogenetics, 6: 1, 1996). When human P450 and rat P450 genes that belong to the same subfamily are compared, differences are observed in properties, and differences are occasionally observed in substrates or metabolites. Thus, information regarding the metabolism of a certain drug in rats is not applicable to humans, and there are needs for development of a test system that can accurately predict drug metabolism in humans (Funae et al., Bioscience & industry, 55: 81, 1997).
Use of human hepatic microsomes is a means for investigating drug metabolism in humans; however, it is difficult to obtain human hepatic microsomes. Meanwhile, genetic engineering techniques have enabled preparation of human enzymes in a relatively easy manner. This enables a stable supply of human enzymes that satisfy the same spec, and use of such techniques is thus taken into consideration (Kamataki, Report from the Biosafety Research Center, Foods, Drugs and Pesticides (An-Pyo Center), 7: 27, 1997). And, in vitro systems for investigating an effect of drugs that have been metabolized by P450 and activated on the living body have been constructed. In such systems, hepatic microsomes and drugs are added to cell cultures to investigate the effect of the metabolites, which had been metabolized outside the cells, on the aforementioned cells. In such a case, activated substances adsorb to cell membranes and only some of such substances can enter into the cells. Accordingly, it is considered that the effect of metabolites on cells cannot be accurately understood by such systems. P450 is considered that, when cells express P450, drugs that had invaded into cells without adsorbing on cell membranes are activated in the cells and the effect of metabolites, including toxicity, is accurately reproduced. Thus, use of cells into which the human P450 gene has been introduced for evaluation of toxicity of metabolites is considered preferable (Kamataki et al., Toxicology Letters, 82-83: 879, 1995).
At present, however, methods involving the use of in vitro expression systems suffer from some drawbacks. The expression system involving the use of yeast cells into which human P450 had been introduced (e.g., Kovaleva et al., Biochem. Biophys. Res. Commun., 221: 129, 1996) is advantageous in that P450 is expressed to some extent without modification of P450 cDNA; however, this system disadvantageously contains P450 of yeast cells. The expression system involving the use of E. coli (e.g., Gillam et al., Arch. Biochem. Biophys., 305: 123, 1993) is easy to handle, and this system can produce a large quantity of enzymes. However, the N-terminal amino acid of P450 to be expressed is required to be modified, in order to stably express P450. This system has drawbacks such that, for example, modification as described above may influence enzyme activity and further addition of reducing enzymes is necessary since E. coli does not have reducing enzymes that are necessary for exhibiting P450 activity. Also, the system involving the use of insect cells and baculoviruses (e.g., Asseffa et al., Arch. Biochem. Biophys., 274: 481, 1989) can express P450 at high levels, and it does not necessitate modification of N-terminal amino acids, although manipulations for expression require some skill. Since the system involving the use of HepG2 cell derived from human hepatic cancer and vaccinia virus (e.g., Shou et al., Mol. Carcinog., 10: 159, 1994) or a system involving use of human B lymphocytes uses human cells, P450 may be expressed in a manner more similar to that in the in vivo environment. When vaccinia virus or HepG2 cell microsome is used, however, attention should be paid to safety (Funae et al., Bioscience & industry, 55: 81, 1997).
Biological roles and regulation of drug-metabolizing enzymes have not yet been fully elucidated. Experimental systems involving the use of animal cells, yeast cells, insect cells, and bacterial cells can function as model systems for investigating the roles of P450 in drug metabolism in vitro and chemical carcinogenesis. However, the fact that such systems do not fully reflect the in vivo conditions because of other factors such as pharmacokinetic parameters should be taken into consideration when using such systems (Wolf et al., J. Pharm. Pharmacol., 50: 567, 1998). If an experimental animal into which the human P450 gene has been introduced and in which the same metabolites as those produced by humans are produced in vivo is developed, toxicity as well as pharmacological effects of metabolites that are generated specifically in humans could be advantageously investigated with the use of animals (Kamataki et al., Yakubutsu Dotai (pharmacokinetics), 13: 280, 1998). To this end, transgenic mice into which the P450 gene had been introduced have been researched. For example, Ramsden et al. (Ramsden et al., J. Biol. Chem., 268: 21722, 1993) constructed transgenic mice into which the rat Cyp2B2 gene had been introduced. It is known that rat Cyp2B2 gene expression is regulated in a tissue-specific and development-specific manner and that such expression is induced by phenobarbital. When inducing expression of a transgene by phenobarbital in transgenic mice, use of an 800-bp promoter sequence alone is insufficient, and use of a gene sequence located upstream is necessary. Also, the control of the transgene expression requires a sequence located several tens of kb upstream of the transcription initiation site, and such sequence may be able to reproduce expression level and tissue specificity (Ramsden et al., J. Biol. Chem., 268: 21722, 1993).
Also, Loefgren et al. constructed transgenic mice comprising bacterial artificial chromosomes (BACs) retaining CYP2C18 or CYP2C19 and reported sexual differences in expression. In this system, the site of gene introduction is mouse chromosome 2 Cl and the copy number was 11-13. Since the copy number of human genes is generally 2, such transgenic mice were found to be insufficient as models for physiologically expressing human CYP2C (Loefgren et al., American Society for Pharmacology and Experimental Therapeutics, 36: 955-962, 2008).
Also, Yu et al. (Yu et al., Endocrinology, 146: 2911, 2005) constructed transgenic mice comprising the bacterial artificial chromosome (BAC) retaining CYP3A4 that is expressed specifically in adult humans. In this example, the introduced CYP3A4 gene was expressed only in 2-week-old and 4-week-old mice; however, gene expression was observed in 8-week-old mice when an expression inducer was administered. These transgenic mice exhibited poor development in the mammary glands, and the survival of progeny thereof was poor. Regarding this system, the site of introduction and the copy number of the introduced genes have not been tested. In order to verify that such poor development or survival is caused by the CYP3A4 gene, accordingly, it was considered to be necessary to investigate reproducibility in mouse lines different in copy numbers and insertion sites.
Further, Li et al. (Li et al., Archs. Biochem. Biophys., 329: 235, 1996) constructed transgenic mice having CYP3A7 which is expressed specifically in human embryos. In this example, a metallothionein promoter was used, and induction of tissue-specific expression of the P450 gene was not observed. Specifically, expression of the introduced CYP3A7 gene in the liver was observed only in one of six transgenic mouse lines, expression of the gene was observed in various organs in other strains, and the native tissue-specificity was not observed. Accordingly, use of the metallothionein promoter may not be sufficient to express a P450 gene in a liver-specific manner.
Regarding CYP3A, application thereof as a tool for research on toxicity during the fetal period has been studied (Kamataki et al., Toxicology Letters, 82-83: 879, 1995). Also, Campbell et al. (Campbell et al., J. Cell Sci., 109: 2619, 1996) constructed transgenic mice into which the gene prepared by linking a promoter sequence of rat Cyp1A1 gene and an upstream sequence thereof to a lacZ gene had been introduced, and analyzed regulation of gene expression by Cyp1A1 using the transgenic mice.
In addition to transgenic mice, P450 knockout mice have been developed, and use of such knockout mice as an important tool for elucidating the influence on development or homeostasis at the cellular level and the roles of P450 regarding in vivo toxicity of drugs or chemical substances is expected (McKinnon et al., Clin. Exp. Pharmacol. Physiol., 25: 783, 1998). For example, two research groups constructed knockout mice lacking endogenous Cyp1a2 (Pineau et al., Proc. Natl. Acad. Sci. U.S.A., 92: 5134, 1995). The Cyp1a2 knockout mice prepared by Pineau et al. (Pineau et al., Proc. Natl. Acad. Sci. U.S.A., 92: 5134, 1995) were normal when the resulting mice were heterozygous; however, they died immediately after birth when they were homozygous. Meanwhile, the Cyp1a2 knockout mice constructed by Liang et al. (Liang et al., Proc. Natl. Acad. Sci. U.S.A., 93: 1671, 1996) did not show any abnormalities in the phenotypes of homozygotes. Such difference is considered to result from different sequences of genes to be deleted. Also, the influence of lacked P450 gene and abnormality of metabolism found using the Cyp1a2 knockout mice have been reported (e.g., Genter et al., Biochem. Pharmacol., 55: 1819, 1998), and the role of Cyp1a2 in the metabolism system has been elucidated with the use of the knockout mice. Knockout mice lacking Cyp2e1, which is known as a major enzyme for metabolizing ethanol, were constructed (Lee et al., J. Biol. Chem., 271: 12063, 1996). Cyp2e1 is known to be involved with metabolism of acetaminophen, acetone, or arachidonic acid, in addition to ethanol. Homozygotes that completely lack Cyp2e1 were not different from wild-type mice in appearance; however, resistance to acetaminophen was improved, and the results of pathological observation suggested that Cyp2e1-mediated metabolism is significantly involved with acetaminophen-induced hepatic toxicity. All of these P450 gene knockout mice were created for the purpose of elucidating functions of such gene by knocking-out the gene of interest, and an increase in the expression level of the introduced foreign P450 gene is not intended.
Meanwhile, Herwaarden et al. produced knockout mice lacking the Cyp3a gene, they further produced mice comprising a human CYP3A4 gene expressed in the liver or small intestine, and they reported research regarding docetaxel metabolism. In these mice, however, the human CYP3A4 gene was ligated to a site downstream of a liver- or small-intestine-specific promoter and forced to express, and thus, such mice would not physiologically reproduce expression levels in humans (Herwaarden et al., J. Clin. Invest., 117: 3583-3592, 2007).
Under such circumstances, for example, WO 01/011951 discloses that a partial fragment of a human normal fibroblast-derived chromosome 7 is introduced into a mouse ES cell (embryonic stem cell) by means of a microcell method, and a chimeric mouse that harbors the human chromosome fragment in normal tissues and expresses a human CYP3A4 gene in the liver and small intestine by induction with a drug is obtained with the use of such ES cells. In this connection, WO 01/011951 discloses the #7-HAC vector obtained by translocating a human chromosome 7 fragment (approximately 5 Mb) comprising the CYP3A gene (hereafter such genes may be referred to as the “CYP3A gene cluster”) to the SC20-HAC vector (FERM BP-7583; JP Patent Publication (kokai) 2005-230020 A), although such vector is incapable of gene transmission to progeny. Further, WO 01/011951 discloses the creation of mice comprising human P450 gene (belonging to CYP3A family) and of mice with disrupted murine endogenous P450 gene (belonging to Cyp3a family).
As disclosed in WO 01/011951, a mouse retaining a partial human chromosome 7 fragment comprising human CYP3A gene or an approximately 5 Mb region of human chromosome 7 comprising human CYP3A gene was introduced into a human artificial chromosome vector (SC20), which is known to be stable in mice, to prepare a mouse retaining the human artificial chromosome vector, although stable transmission of the gene from a chimeric mouse to progeny was impossible. If a mouse that can transmit a gene to progeny cannot be obtained, then a mouse must be produced from a chimeric mouse, indicating that embryo manipulation is necessary each time and mice having homogeneous genetic background cannot be obtained. Further, this means that progeny mice into which a plurality of human P450 genes of interest have been introduced and in which endogenous drug-metabolizing enzymes have been disrupted cannot be obtained. As a cause that the human CYP3A genes are not transmitted from a chimeric mouse retaining the human CYP3A genes disclosed in WO 01/011951 to progeny, it has been reported that overexpression of genes involved in genomic imprinting or genes that are important for development and germ cell differentiation would lead to embryonic lethality (Okita et al., Genomics., 81 (6): 556, 2003; Sun F L, Dean W L, Kelsey G, Allen N D, Reik W.: Transactivation of Igf2 in a mouse model of Beckwith-Wiedemann syndrome, Nature, 1997 Oct. 23; 389 (6653): 785, 787; and Puech et al., Proc. Natl. Acad. Sci. U.S.A., 97: 10090, 2000).
In contrast, the human artificial chromosome vector (which, hereafter, may be referred to as the “HAC vector”) is advantageous in that: for example, 1) it is independently maintained without being inserted into the host chromosome and it thus does not disrupt host genes; 2) it is stably retained at a given copy number, it is influenced by physiological expression control of a host cell, and neither overexpression of an introduced gene nor lost expression of the gene is caused; and 3) the size of a DNA that can be introduced is not restricted, and, thus, a gene containing an expression control region or a plurality of genes/isoforms can be introduced. As described above, because the human artificial chromosome vector has advantages that conventional vectors (i.e., virus, YAC, BAC, PAC, cosmid, and plasmid vectors) do not have, the human artificial chromosome vector is expected to function as a vector used for analyzing functions of novel genes or as a system for creating a human-type animal model (e.g., Kuroiwa et al., Nature Biotech., 18: 1086, 2000; and Tomizuka et al., Proc. Natl. Acad. Sci. U.S.A., 97: 722, 2000).